We have been using an autotransporter produced by E. coli O157:H7 called EspP as a model protein to study autotransporter biogenesis. In one line of investigation we have been examining the mechanism by which the EspP passenger domain is translocated across the OM. It was originally proposed that the passenger domain is secreted through a channel formed by the covalently linked beta domain (whence the name autotransporter), but results that we obtained from both biochemical and structural studies are inconsistent with this hypothesis. We found that the insertion of a small linker into the EspP passenger domain effectively creates a translocation intermediate by transiently stalling translocation near the site of the insertion. By using a site-specific photocrosslinking approach we found that residues adjacent to the stall point interact with BamA, a component of a heterooligomeric complex (Bam complex) that catalyzes OM protein assembly, and that residues that are trapped in the periplasm interact with the periplasmic chaperones SurA and Skp. The EspP-BamA interaction was short-lived and could only be detected when passenger domain translocation was stalled. These results support a model in which molecular chaperones prevent misfolding of the passenger domain prior to its secretion and the Bam complex plays a major role in facilitating both the integration of the beta domain into the OM and the translocation of the passenger domain across the OM. Recently, we found that periplasmic chaperones and specific components of the Bam complex interact with the EspP beta domain in a temporally and spatially regulated fashion. While the chaperone Skp initially interacted with the entire beta domain, BamA, BamB and BamD subsequently interacted with discrete beta domain regions. BamB and BamD remained bound to the beta domain longer than BamA and therefore appeared to function at a later stage of assembly. Our results suggest that the hitherto enigmatic BamB and BamD proteins play a direct role in the membrane integration of autotransporter beta domains and possibly other beta barrel proteins. Interestingly, we also obtained evidence that the completion of beta domain assembly is regulated by an intrinsic checkpoint mechanism that requires the completion of passenger domain secretion. In a second line of investigation, we have been examining the energetics of passenger domain secretion. Although passenger domain secretion does not appear to use ATP, the energy source for this reaction is unknown. We found that efficient secretion of the EspP passenger domain requires the stable folding of a C-terminal 17 kD passenger domain segment. Because translocation proceeds in a C-to-N-terminal direction, this is the first segment of the passenger domain that is exposed on the cell surface. We found that mutations that perturb the folding of the C-terminal segment do not affect its translocation across the OM, but impair the secretion of the remainder of the passenger domain. Interestingly, an examination of kinetic folding mutants strongly suggested that the 17 kD segment folds in the extracellular space. Our results provide the first direct evidence that the vectorial folding of a protein can act as a Brownian ratchet that drives its translocation across a biological membrane.